US4865994A - Detection method for amino acid derivatives - Google Patents

Detection method for amino acid derivatives Download PDF

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US4865994A
US4865994A US07/185,324 US18532488A US4865994A US 4865994 A US4865994 A US 4865994A US 18532488 A US18532488 A US 18532488A US 4865994 A US4865994 A US 4865994A
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phenylthiocarbamyl
amino acid
reacting
amino
thiazolinone
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Akira Tsugita
Isamu Arai
Tatsuaki Ataka
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Seiko Instruments Inc
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Seiko Instruments Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/534—Production of labelled immunochemicals with radioactive label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6806—Determination of free amino acids
    • G01N33/6809—Determination of free amino acids involving fluorescent derivatizing reagents reacting non-specifically with all amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6818—Sequencing of polypeptides
    • G01N33/6824—Sequencing of polypeptides involving N-terminal degradation, e.g. Edman degradation
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/141111—Diverse hetero atoms in same or different rings [e.g., alkaloids, opiates, etc.]

Definitions

  • This invention relates to a method for detecting amino acids for application to sequence determination from the amino (N) terminal of a protein. More particularly, it relates to a method for detecting amino acids with high sensitivity by using an amino compound containing a radioactive iodine isotope, for example, iodohistamine or a fluorescent amino compound, for example, aminopyrene or aminofluorene.
  • a radioactive iodine isotope for example, iodohistamine or a fluorescent amino compound, for example, aminopyrene or aminofluorene.
  • radioactive isotope derivatives take part in the main reaction in the Edman degradation method. If high radioactivity is used for the purpose of realizing high sensitivity, not only radioactive disintegration increases, which adversely affects a yield itself in the Edman amino acid sequence determination, but also the contamination of the environment occurs.
  • the present inventive method comprises reacting thiazolinone derivatives with a radioactive isotope labelled amine compound or fluorescent amino compound to form phenylthiocarbamyl amino acid derivatives and detecting these amino acid derivatives with high sensitivity by means of a radioactivity detector or a fluorescence spectrophotometer.
  • FIG. 1 is a process flowsheet showing the detection method of this invention.
  • FIG. 2 is a process flowsheet of a prior art method.
  • FIG. 3 are HPLC charts of a reaction mixture and a reaction product (mixing or reaction between ATZ-Leu (the anilinothiazolinone derivative of leucine) and iodohistamine).
  • FIG. 4 is a thin-layer chromatogram of various iodohistamine derivatives of PTC amino acids.
  • FIG. 5 are HPLC charts of a reaction mixture and a reaction product (mixing or reaction between ATZ-Leu and aminofluorene)
  • This example demonstrates a basic detection method wherein 5-thiazolinone derivatives of amino acids (ATZ) were reacted with a radioactive iodine isotope-labelled iodohistamine to form phenylthiocarbamyl amino acid derivatives and these products were detected with high sensitivity.
  • Leu-Ala (202 mg) was dissolved in 50% aqueous pyridine, and the pH was adjusted to 8.6 by the addition of 2N NaOH. Subsequently, PITC was added thereto. The pH was kept at 8.6 by the addition of 2N NaOH because it was decreasing with the addition of PITC. After the variation in the pH had been stopped, the solution was heated at 40° C. for one hour. After the reaction, the reaction solution was washed with benzene. After the benzene dissolved in the water phase had been purged with N 2 gas, the pH was adjusted to 2 by the addition of 1N HCl, whereupon PTC-Leu-Ala was obtained in the form of a white precipitate.
  • PITC-Leu-Ala 100 mg was dissolved in TFA (1 ml), and the solution was heated at 50° C. for 5 min. After the reaction, the solution was evaporated to dryness. Butyl chloride was added to the residue to thoroughly dissolve the product in it, and the resulting solution was passed through a cellulose column (Ala was adsorbed on the column). The effluent was collected and evaporated to dryness. ATZ-Leu was obtained in the form of a white solid.
  • reaction formula shows the amino group of iodohistamine attacks the carbonyl group of ATZ amino acid to give a product.
  • the reaction is the reverse of a reaction for cleaving the peptide bond between PTC amino acid and the peptide.
  • ATZ-Leu (70 mg) obtained from PTC-Leu-Ala was dissolved in 30% pyridine/dimethylformamide (20 ml), to which was then added iodohistamine 2HCl (200 mg), the resulting mixture was heated with agitation at 50° C. for one hour.
  • the reaction solution was evaporated to dryness and the residue was dried to solid and extracted with 1M sodium bicarbonate and ethyl acetate.
  • the ethyl acetate phase was dried and evaporated to dryness.
  • the residue was recrystallized from benzene to obtain a product in the form of a white crystal.
  • FIG. 3 (i) shows an HPLC chromatogram just after mixing
  • FIG. 3 (ii) shows an HPLC chart after heating at 50° C. for 30 min
  • FIG. 3 (iii) shows an HPLC chart after heating at 50° C. for one hour.
  • the coupling reaction between the thiazolinone derivative of leucine and iodohistamine was completed by heating at 50° C. for one hour, so that the thiazolinone derivative was detected in the form of a PTC leucine derivative.
  • SERVACHROM Packing (SERVACHROM is a tradename of SERVA FEINBIOCHAMICA GmbH for high pressure liquid chromatography columns)
  • This example demonstrates one wherein a method similar to that described in Example 1 was applied to various dipeptides and a pentapeptide, and the detection was performed by using thin-layer chromatography and an X-ray film.
  • the dipeptides and pentapeptide used were as follows: Leu-Gly, Pro-Phe, Phe-Ala, Met-Leu, Ser-Phe, Ile-Ser, Gly-Glu, Ala-Ala, Val-Glu, Gln-Gly, and Tyr-Gly-Gly-Phe-Leu.
  • Example 2 A similar method to that described in Example 1 was followed. Namely, each of the above peptides was reacted with phenyl isothiocyanate (PITC) to form a phenylthiocarbamyl peptide, which was converted into a thiazolinone derivative by cyclization and scission by treatment with trifluoroacetic acid. This thiazolinone derivative was reacted with iodohistamine labelled with a radioactive isotope to form a phenylthiocarbamylamino acid derivative.
  • PITC phenyl isothiocyanate
  • phenylthiocarbamyl amino acid derivatives were developed on a thin-layer chromatogram and then detected as clear exposed spots on an X-ray film after exposure for several hours.
  • a solvent system comprising benzene, methanol and tert-butyl alcohol in a volume ratio of 8:1:1 was used in development.
  • Example 2 A similar procedure to that described in Example 1 was followed till a step of obtaining anilino thiazolinone derivatives.
  • the anilino thiazolinone derivative of leucine hereinafter abbreviated as ATZ-Leu was used.
  • reaction was performed in the following way. To ATZ-Leu (70 mg, 0.21 mM) was added 9-aminofluorene (130 mg, 0.6 mM) dissolved in 30% pyridine-dimethylformamide, and the resulting mixture was reacted by heating at 50° C. for one hour.
  • FIG. 5 (i) shows an HPLC chart of the reaction mixture just after mixing
  • FIG. 5 (ii) shows an chromatogram chart after reacting by heating at 50° C. for one hour.
  • anilinothiazolinone derivative (a) of leucine after the reaction was detected in the form of a phenylthiocarbamyl leucine derivative (c).
  • a comparison of the sensitivity of detection between the method this invention and of the prior art phenylthiohydantoin amino acid detection method revealed that the former was extremely high as follows:
  • the gist of this invention resides in reacting an iodine-labelled amine compound or a fluorescent amino compound with a thiazolinone derivative of an amino acid to form a phenylthiocarbamyl amino acid derivative and detecting it with high sensitivity.
  • a thiazolinone derivative of an amino acid to form a phenylthiocarbamyl amino acid derivative and detecting it with high sensitivity.

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Abstract

A method for detecting amino acid derivatives in which 2-anilino-5-thiazolinone derivatives of amino acids are reacted with radioactive iodine siotope-labeled amino compounds or with fluorescent amino compounds to form phenylthiocarbamyl amino acid derivatives. The phenylthiocarbamyl amino acid derivatives are detected with a high level of sensitivity by a radioactive detector or a fluorescence spectrophotometer.

Description

This is a Rule 62 continuation application of parent application Ser. No. 865,076, filed May 19, 1986, now abandoned.
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a method for detecting amino acids for application to sequence determination from the amino (N) terminal of a protein. More particularly, it relates to a method for detecting amino acids with high sensitivity by using an amino compound containing a radioactive iodine isotope, for example, iodohistamine or a fluorescent amino compound, for example, aminopyrene or aminofluorene.
2. Description of the Prior Art
For the detection of amino acids in the final step of the phenylisothiocyanate method according to Edmon, P., Acta. Chem. Scand. 10 761 (1956), which is an N-terminal sequence determination method, it has been a usual practice as shown in FIG. 2 that thiazolinone derivatives are treated with an acid to form phenylthiohydantoin (PTH) derivatives and these derivatives are determined spectrophotometrically.
Although the prior art method for spectrophotometrically detecting PTH derivatives is simple and convenient as detection means, it cannot fully cope with a recent trend toward more highly sensitive analysis of a protein with a smaller amount of specimen.
For the high sensitivity detection of amino acids, methods of using 32 S PITC labelling or 135 I PITC labelling are shown in the following publication, PITC standing for phenylisothiocyanate:
W. G. Lauer, Fundamental Techniques in Virology, Eds. K. Habel and N. P. Salgman p. 379 (1969) Academic Press N.Y.
C. J. Burrell, P. D. Cooper, J. M. Swann, Aust J. Chem. 28 2289, (1975).
In above methods, radioactive isotope derivatives take part in the main reaction in the Edman degradation method. If high radioactivity is used for the purpose of realizing high sensitivity, not only radioactive disintegration increases, which adversely affects a yield itself in the Edman amino acid sequence determination, but also the contamination of the environment occurs.
SUMMARY OF THE INVENTION
The present inventive method comprises reacting thiazolinone derivatives with a radioactive isotope labelled amine compound or fluorescent amino compound to form phenylthiocarbamyl amino acid derivatives and detecting these amino acid derivatives with high sensitivity by means of a radioactivity detector or a fluorescence spectrophotometer.
Therefore, it is an object of this invention to provide a novel method for detecting amino acids with higher sensitivity.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a process flowsheet showing the detection method of this invention.
FIG. 2 is a process flowsheet of a prior art method.
FIG. 3 are HPLC charts of a reaction mixture and a reaction product (mixing or reaction between ATZ-Leu (the anilinothiazolinone derivative of leucine) and iodohistamine).
FIG. 3 (i): just after mixing
FIG. 3 (ii): after heating at 50° C. for 30 min
FIG. 3 (iii): after heating at 50° C. for one hour
(a) thiazoline derivative of leucine
(b) phenylthiocarbamyl (PTC) derivative of leucine
FIG. 4 is a thin-layer chromatogram of various iodohistamine derivatives of PTC amino acids.
FIG. 5 are HPLC charts of a reaction mixture and a reaction product (mixing or reaction between ATZ-Leu and aminofluorene)
DETAILED DESCRIPTION OF THE INVENTION
This invention will now be described in more detail with reference to examples.
EXAMPLE 1
This example demonstrates a basic detection method wherein 5-thiazolinone derivatives of amino acids (ATZ) were reacted with a radioactive iodine isotope-labelled iodohistamine to form phenylthiocarbamyl amino acid derivatives and these products were detected with high sensitivity.
This method will be described in the following order. Hereinbelow, a dipeptide (Leu-Ala) consisting of leucine (Leu) and alanine (Ala) is used instead of a protein for simplicity.
1. PTC-Leu-Ala and ATZ-Leu
(i) synthesis of PTC-Leu-Ala
(ii) synthesis and purification of ATZ-Leu
2. coupling reaction
3. detection of phenylthiocarbamyl amino acid derivatives
1. PTC-Leu Ala and ATZ-Leu
(i) synthesis of PITC-Leu-Ala ##STR1##
Leu-Ala (202 mg) was dissolved in 50% aqueous pyridine, and the pH was adjusted to 8.6 by the addition of 2N NaOH. Subsequently, PITC was added thereto. The pH was kept at 8.6 by the addition of 2N NaOH because it was decreasing with the addition of PITC. After the variation in the pH had been stopped, the solution was heated at 40° C. for one hour. After the reaction, the reaction solution was washed with benzene. After the benzene dissolved in the water phase had been purged with N2 gas, the pH was adjusted to 2 by the addition of 1N HCl, whereupon PTC-Leu-Ala was obtained in the form of a white precipitate.
Yield: 270 mg, % yield: 80%, m.p.: 148° C.
(ii) synthesis and purification of ATZ-Leu ##STR2##
The above PITC-Leu-Ala (100 mg) was dissolved in TFA (1 ml), and the solution was heated at 50° C. for 5 min. After the reaction, the solution was evaporated to dryness. Butyl chloride was added to the residue to thoroughly dissolve the product in it, and the resulting solution was passed through a cellulose column (Ala was adsorbed on the column). The effluent was collected and evaporated to dryness. ATZ-Leu was obtained in the form of a white solid.
Yield: 70 mg, % yield: 95%.
2. coupling reaction ##STR3##
As the above reaction formula shows the amino group of iodohistamine attacks the carbonyl group of ATZ amino acid to give a product. The reaction is the reverse of a reaction for cleaving the peptide bond between PTC amino acid and the peptide.
ATZ-Leu (70 mg) obtained from PTC-Leu-Ala was dissolved in 30% pyridine/dimethylformamide (20 ml), to which was then added iodohistamine 2HCl (200 mg), the resulting mixture was heated with agitation at 50° C. for one hour. The reaction solution was evaporated to dryness and the residue was dried to solid and extracted with 1M sodium bicarbonate and ethyl acetate. The ethyl acetate phase was dried and evaporated to dryness. The residue was recrystallized from benzene to obtain a product in the form of a white crystal.
Yield: 120 mg, % yield: 83%.
There was good agreement between the results by elementary analysis of this product and the values calculated from its theoretical formula.
3. detection of phenylthiocarbamyl amino acid derivatives
The reaction mixture including the reaction product was chromatographed by high-pressure liquid chromatography (HPLC) and the detection was made by ultraviolet spectra (at 269 nm). The results are shown in FIG. 3. FIG. 3 (i) shows an HPLC chromatogram just after mixing, FIG. 3 (ii) shows an HPLC chart after heating at 50° C. for 30 min, and FIG. 3 (iii) shows an HPLC chart after heating at 50° C. for one hour. As these figures clearly show, the coupling reaction between the thiazolinone derivative of leucine and iodohistamine was completed by heating at 50° C. for one hour, so that the thiazolinone derivative was detected in the form of a PTC leucine derivative.
(conditions of HPLC)
column:
SERVA Cat. No. 4231
250 mm×4.6 mm
SERVACHROM Packing (SERVACHROM is a tradename of SERVA FEINBIOCHAMICA GmbH for high pressure liquid chromatography columns)
S:100:polyol:RP18
5 μm
solvent system:
______________________________________                                    
buffer solution                                                           
               organic solvent                                            
______________________________________                                    
0.015 M        CH.sub.3 CN--MeOH                                          
NaOAc (pH = 5                                                             
with AcOH)     (4:1)                                                      
1              5                                                          
______________________________________
effluent flow rate: 1.5 ml/min
detection: 269 nm
chart speed: 10 mm/min
NaOAc=sodium acetate; AcOH=acetic acid
EXAMPLE 2
This example demonstrates one wherein a method similar to that described in Example 1 was applied to various dipeptides and a pentapeptide, and the detection was performed by using thin-layer chromatography and an X-ray film.
The dipeptides and pentapeptide used were as follows: Leu-Gly, Pro-Phe, Phe-Ala, Met-Leu, Ser-Phe, Ile-Ser, Gly-Glu, Ala-Ala, Val-Glu, Gln-Gly, and Tyr-Gly-Gly-Phe-Leu.
A similar method to that described in Example 1 was followed. Namely, each of the above peptides was reacted with phenyl isothiocyanate (PITC) to form a phenylthiocarbamyl peptide, which was converted into a thiazolinone derivative by cyclization and scission by treatment with trifluoroacetic acid. This thiazolinone derivative was reacted with iodohistamine labelled with a radioactive isotope to form a phenylthiocarbamylamino acid derivative. A variety of the formed phenylthiocarbamyl amino acid derivatives were developed on a thin-layer chromatogram and then detected as clear exposed spots on an X-ray film after exposure for several hours. In the thin-layer chromatography, a solvent system comprising benzene, methanol and tert-butyl alcohol in a volume ratio of 8:1:1 was used in development.
The results are shown in FIG. 4, wherein ordinates represent RF (retention factor) values. As compared with the sensitivity of detection of the prior art, that of this invention was extremely high as follows:
______________________________________                                    
detection method      sensitivity of detection                            
______________________________________                                    
(prior art) phenylthiohydantoinamino                                      
acid method           ˜10                                           
                               pico mole                                  
this invention        01 ˜ 10                                       
                               femto mole                                 
______________________________________
In this Example, no radioactive substance is used in the main reaction, though it is used in only the final step in the sequence determination method, so that the main reaction is quite the same as in the prior art and a radioactive substance is used in a special vessel in only the final modification reaction of a reaction product intermediate (ATZ) in each step and therefore the reaction itself does not differ at all from that in the prior art. Therefore, it is possible to perform the reaction without adversely affecting the reaction yield while minimizing radioactive contamination.
EXAMPLE 3
This example demonstrates wherein 5-anilino 5-thiazolinone derivatives of amino acids were reacted with a fluorescent amino compound (9-aminofluorene) to form phenylthiocarbamyl amino acid derivatives, and the products were detected with high sensitivity.
A similar procedure to that described in Example 1 was followed till a step of obtaining anilino thiazolinone derivatives. The anilino thiazolinone derivative of leucine (hereinafter abbreviated as ATZ-Leu) was used.
This reaction proceeded according to the following reaction formula to form a phenylthiocarbamyl amino acid derivative. ##STR4##
The reaction was performed in the following way. To ATZ-Leu (70 mg, 0.21 mM) was added 9-aminofluorene (130 mg, 0.6 mM) dissolved in 30% pyridine-dimethylformamide, and the resulting mixture was reacted by heating at 50° C. for one hour.
After the reaction, the reaction solution was evaporated to dryness. CHCl3 was added to the residue and the insoluble matter (9-aminofluorene.HCl) was separated by filtration. The CHCl3 was washed with 0.1N HCl and 0.11N NaHCO3, over Na2 SO4 and concentrated to dryness. The residue was recrystallized from benzene to obtain a product in the form of a white crystal.
Yield: 130 mg, % yield: 88%, m.p.: 225° C.
Each of the reaction mixture and the reaction product was passed through a high-pressure liquid chromatography (HPLC) column, and the detection was performed by ultraviolet fluorescence spectra (at 269 nm). The results are shown in FIG. 5. FIG. 5 (i) shows an HPLC chart of the reaction mixture just after mixing, and FIG. 5 (ii) shows an chromatogram chart after reacting by heating at 50° C. for one hour. As these figures clearly show, anilinothiazolinone derivative (a) of leucine after the reaction was detected in the form of a phenylthiocarbamyl leucine derivative (c). In addition, a comparison of the sensitivity of detection between the method this invention and of the prior art phenylthiohydantoin amino acid detection method revealed that the former was extremely high as follows:
______________________________________                                    
detection method      sensitiviy of detection                             
______________________________________                                    
Phenylthiohydantoin amino acid                                            
method (prior art)    ˜10                                           
                               pico mole                                  
this invention        0.1 ˜ 1                                       
                               pico mole                                  
______________________________________
Extremely high sensitivity of detection of the amino acid derivatives of this invention is shown below by comparison with the sensitivity of detection in the prior art method in which a phenylthiocarbamyl amino acid derivative (phenylthiohydantoin amino acid) is used:
______________________________________                                    
detection method      sensitivity of detection                            
______________________________________                                    
phenylthiohydantoin amino acid                                            
detection method (prior art)                                              
                      1 ˜ 10                                        
                               pico mole                                  
this invention:                                                           
when iodine isotope labelling is used                                     
                      1 ˜ 10                                        
                               femto mole                                 
when a fluorescent                                                        
amino compound is used                                                    
                      0.1 ˜ 1                                       
                               pico mole.                                 
______________________________________
The gist of this invention resides in reacting an iodine-labelled amine compound or a fluorescent amino compound with a thiazolinone derivative of an amino acid to form a phenylthiocarbamyl amino acid derivative and detecting it with high sensitivity. Although only those examples in which iodohistamine or aminofluorene was used were shown above, it is apparent that the effect of this invention can be attained by using other iodine-labelled or fluorescent amino compounds and is not limited to particular examples. Therefore, the detection method for amino acids of this invention is of great industrial value.

Claims (8)

What is claimed is:
1. A method for detecting 5-thiazolinone derivatives of amino acids comprising:
reacting (a) 5-thiazolinone derivatives of amino acids with (b) an amino compound labeled with a radioactive isotope to form (c) phenylthiocarbamyl amino acid derivatives according to the reaction scheme: ##STR5## wherein X is a residue of a radioactive isotope label and R1 is hydrogen or a hydrocarbon radical; and
detecting the phenylthiocarbamyl amino acid derivatives.
2. A method of detecting amino acids comprising:
reacting (d) phenylisothiocyanate with (e) a protein or peptide to form (f) phenylthiocarbamyl protein;
reacting the (f) phenylthiocarbamyl protein with (g) an acid in an anhydrous condition to effect cyclization and scission and to form (a) a 5-thiazolinone derivative; and
reacting the (a) 5-thiazolinone derivative with (b) an amino compound labeled with a radioactive isotope to form (c) phenylthiocarbamyl amino acid derivatives according to the reaction scheme: ##STR6## wherein each of R1, R2, and R3 is hydrogen or a hydrocarbon radical, X is a residue of radioactive isotope label, and wherein (h) is the cleaved peptide or protein.
3. A method for detecting 5-thiazolinone derivatives of amino acids comprising:
reacting (a) 5-thiazolinone derivatives of amino acids with (b) a fluorescent amino compound to form (c) phenylthiocarbamyl amino acid derivatives according to the reaction scheme: ##STR7## wherein X is a fluorescent compound and R1 is hydrogen or a hydrocarbon radical.
4. A method of detecting amino acids comprising:
reacting (d) phenylisothiocyanate with (e) a protein or peptide to form (f) phenylthiocarbamyl amino;
reacting the (f) phenylthiocarbamyl protein with (g) an acid in an anhydrous condition to effect cyclization and scission and to form (a) a 5-thiazolinone derivative; and
reacting the (a) 5-thiazolinone derivative with (b) a fluorescent amino compound to form (c) phenylthiocarbamylamino acid derivatives according to the reaction scheme: ##STR8## wherein each of R1, R2, R3 is hydrogen or a hydrocarbon radycal, X is a fluorescent compound, and wherein (h) is the cleaved peptide or protein.
5. A method for detecting 5-thiazolinone derivatives of amino acids comprising:
reacting (a) 5-thiazolinone derivatives of amino acids with (b) an amino compound labeled with a radioactive isotope to form (c) phenylthiocarbamyl amino acid derivatives according to the reaction scheme: ##STR9## wherein X is a residue of a radioactive isotope label and R1 is an amino acid side chain; and
detecting the phenylthiocarbamyl amino acid derivatives.
6. A method of detecting amino acids comprising:
reacting (d) phenylisothiocyanate with (e) a protein or peptide to form (f) phenylthiocarbamyl protein;
reacting the (f) phenylthiocarbamyl protein with (g) an acid in an anhydrous condition to effect cyclization and scission and to form (a) a 5-thiazolinone derivative; and
reacting the (a) 5-thiazolinone derivative with (b) an amino compound labeled with a radioactive isotope to form (c) phenylthiocarbamyl amino acid derivatives according to the reaction scheme: ##STR10## wherein each of R1, R2, and R3 is an amino acid side chain, X is a residue of a radioactive isotope label, and wherein (h) is the cleaved peptide or protein.
7. A method for detecting 5-thiazolinone derivatives of amino acids comprising:
reacting (a) 5-thiazolinone derivatives of amino acids with (b) a fluorescent amino compound to form (c) phenylthiocarbamyl amino acid derivatives according to the reaction scheme: ##STR11## wherein X is a fluorescent compound and R1 is an amino acid side chain.
8. A method of detecting amino acids comprising:
reacting (d) phenylisothiocyanate with (e) a protein or peptide to form (f) phenylthiocarbamyl protein;
reacting the (f) phenylthiocarbamyl protein with (g) an acid in an anhydrous condition to effect cyclization and scission and to form (a) a 5-thiazolinone derivative; and
reacting the (a) 5-thiazolinone derivative with (b) a fluorescent amino compound to form (c) phenylthiocarbamyl amino acid derivatives according to the reaction scheme: ##STR12## wherein each of R1, R2 and R3 is an amino acid side chain, X is a fluorescent compound, and wherein (h) is the cleaved peptide or protein.
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JPS61264264A (en) 1986-11-22
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EP0202894A2 (en) 1986-11-26
DE3683916D1 (en) 1992-03-26

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